CN103642757A - Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications - Google Patents
Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications Download PDFInfo
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Abstract
The invention discloses a kind of bluetongue virus (BTV) NS2 protein monoclonal antibody BTV-4D4, a B cell epitope recognized thereby and applications. BALB/c mice are subjected to totivirus immunization by utilization of bluetongue virus serotype 8 type (BTV8), splenic lymphocytes of the mice after immunization and SP2/0 cells are fused. A hybridoma cell strain BTV-4D4 secreting anti-BTV-NS2 protein monoclonal antibodies steadily is obtained after screening with BTV 8 as a coating antigen through an indirect ELISA method. The monoclonal antibodies secreted from the hybridoma cell strain and the BTV-NS2 protein are subjected to a specific reaction. The B cell epitope recognized by BTV-4D4 is 149RSNYDV154 after identification by utilization of a phage display technology. The monoclonal antibody and the BTV-NS2 protein B cell epitope recognized by the monoclonal antibody can be used for diagnosis or prevention of bluetongue virus infection.
Description
Technical field
The present invention relates to the monoclonal antibody of a strain of hybridoma strain and secretion thereof, relate in particular to a strain and secrete the hybridoma cell strain of anti-BTV-NS2 protein monoclonal antibody and secreted monoclonal antibody thereof; The invention still further relates to a B cell epitope, relate in particular to the BTV-NS2 protein B cell epitope polypeptide of being identified by said monoclonal antibody; The invention still further relates to the application in preparation diagnosis or prevention BTV infection of above-mentioned hybridoma cell strain, monoclonal antibody and B cell epitope polypeptide, the invention belongs to the field anti-processed of bluetongue.
Background technology
Bluetongue (Bluetongue, BT) is the ruminating animal arthropod borne infection being caused by the blue tongue virus of Reoviridae Orbivirus (Bluetongue virus, BTV).BTV can infect most of domestic and wild ruminating animals, because the susceptibility differing appearance of animal goes out different clinical symptom.The differential mortality of different Prevalent district susceptible animal is very large, generally all can reach 30%, even higher in certain areas.Due to the impact of BT on world economy, OIE (OIE) classifies BT as legal circular disease, and China is a class animal epidemic by its delimitation.The loss of related economic that BT causes is on the one hand by directly reducing the throughput of animal and increasing the mortality ratio of animal, the more important thing is owing to controlling flowing, control the outlet of ox seminal fluid and implementing the required expense of these measure of control and the animal trade loss that indirectly causes of animal.
BT, early than the sheep that is found in South Africa for 1876, is named as " anti-malarial sheep catarrhal fever " for 1902, and 1905 is " bluetongue " by definite designation.20 beginnings of the century, because the introduction of hypersusceptible non-native sheep variety causes BT to start to spread in Africa.BT is considered to a kind of endemicity disease, and America, Africa, Asia, Australia of being positioned at 40 ° of S of latitude and 53 ° of N are hotspots, only within 1996, causes financial loss in global range up to 3,000,000,000 U.S.s.Within 1979, China finds bluetongue at Sheep from Yunnan first, all detects the positive domestic animal of BT subsequently, as Shanxi (1991), Gansu (1990), Sichuan (1988), Anhui (1985), Hubei (1983) etc. in 29 provinces.Since 2006, along with climate warming, BT especially BTV8 in Europe, many countries, as the outburst such as Belgian, Dutch, French, German, have caused serious financial loss, also expanded traditional distribution range of BTV simultaneously.Qin Shaomin etc. (2011) have carried out serosurvey to the regional goat BT in 11, Guangxi, and result shows that positive rate is 6.3%-45.1%, and average positive rate is 20.5%.
BTV serotype is numerous, has found at present 24 serotypes, and along with the new serotype of climate change constantly occurs, as the BTV25(Switzerland being in succession separated to again recently, 2008 years) and BTV26(Kuwait, 2011 years).Yet because cross immunity protectiveness between each serotype is poor, make that vaccine immunity is intricate can not play a very good protection, up to the present not yet have effective vaccine.Early stage Accurate Diagnosis, early prevention, is the most effectual way that prevents the outburst of this disease.So, must strengthen the research of the related work of China BT, carry out necessary tachnical storage.
BTV belongs to Reoviridae (Reoviridae) Orbivirus (Orbivirus), and its genome is segmented linear double-stranded RNA, comprises 10 sections, and size is about 19kb, the 11 kinds of albumen of encoding altogether.By the NS2 of S8 genes encoding, long 357 amino acid, are unique a kind of phosphorylated proteins.NS2 virus copy with assembling process in play an important role, NS2 can be in conjunction with viral single stranded RNA, can be hydrolyzed ribonucleoside triphosphote is nucleoside monophosphate.NS2 is the main component of BTV cells infected viral inclusion body (VIB), great expression in cell.Therefore, by block the one or more function of NS2 in virus replication, virus can not effectively be copied, thereby provide provide protection to body.In addition, to the detection of NS2 antigen, can distinguish natural infection and vaccine inoculation animal.So, filter out the hybridoma cell strain that a strain can secreting specificity antibody, and identify the BTV-NS2 protein-specific B cell epitope that its secreted monoclonal antibody identifies BTV diagnosis or prevention are had to vital role, and lay a good foundation for the development of BTV Epitope tag vaccine.
Summary of the invention
One of the object of the invention is to provide the hybridoma cell strain of a strain secretion BTV-NS2 protein monoclonal antibody;
Two of the object of the invention is to provide a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain, and this monoclonal antibody can specific reaction occur with 24 serotypes B TV;
Three of the object of the invention is to identify the specific b cells epi-position of a BTV-NS2 albumen.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention utilizes BTV8 totivirus as immunogen, and immune BALB/c mouse is got its splenocyte and SP2/0 myeloma cell and merged.In addition, the present invention also utilizes BTV8 totivirus as detection antigen, setting up indirect ELISA detection method screens positive hybridoma cell, the final hybridoma cell strain that obtains the anti-BTV-NS2 protein monoclonal antibody of a strain stably excreting, its culture presevation number is: CGMCC No.7001, called after BTV-4D4, Classification And Nomenclature is: the hybridoma cell strain of secreting anti-BTV-4D4 monoclonal antibody; The preservation time: on December 11st, 2012; Depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.
It is a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain that the present invention also provides, its called after BTV-4D4, Western blot detected result show BTV-4D4 can with BTV-NS2 albumen generation specific reaction, and with contrast BHK-21 cell and do not react.
The present invention utilizes the B cell epitope of the BTV-NS2 albumen that display technique of bacteriophage and pepscan identify BTV-4D4 to identify, this epi-position is accurately orientated as the most at last:
149rSNYDV
154(shown in SEQ ID NO.1).Meanwhile, sequence alignment result shows
149rSNYDV
154in different serotypes BTV, it is high conservative.
Therefore, the present invention proposes the application of described hybridoma cell strain in preparation diagnosis or prevention BTV virus infective medicament.And
The application of described monoclonal antibody in system diagnosis or prevention BTV1 virus infective medicament.And
The linear B cell epitope polypeptide of described BTV-4D4 albumen
149rSNYDV
154(shown in SEQ ID NO.1) application in preparation diagnosis BTV infection medicine.
In sum, the present invention prepares and has identified the conservative B cell epitope of BTV-NS2 prion specificity that a species specificity is identified for monoclonal antibody and this monoclonal antibody of BTV-NS2 albumen, and the conservative B cell epitope of BTV-NS2 prion specificity that monoclonal antibody of the present invention and this monoclonal antibody are identified is that the functional study of setting up BTV immunological detection method and BTV-NS2 albumen is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the reactive result of application Western blot test detection monoclonal antibody BTV-4D4 and BTV-NS2 albumen and BHK-21 cell;
Reacting of the BHK-21 lysis precipitation that 1:BTV-4D4 infects with BTV8; 2:BTV-4D4 reacts with BHK-21 lysis precipitation; M: standard protein Marker
Fig. 2 be phage clone sequencing result and with the comparison of BTV-NS2 protein similar;
Fig. 3 is synthesized small peptide and the reactive indirect ELISA analysis of BTV-4D4 in table 1;
Fig. 4 by conservative property and the specificity analyses of evaluation B cell epitope.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
Main experiment material and source
1. cell is with viral
BHK-21 cell, SP2/0 myeloma cell and BTV1-24 type strain You Zhe research department preserve.
2. main agents
Foetal calf serum, DMEM substratum are purchased from GIBCO company; 50%PEG, 50 * HAT, 50 * HT, FITC mark sheep anti-mouse igg antibody are purchased from Sigma company; The goat dynamics of diaminobenzidine (DAB) colouring reagents box, horseradish peroxidase (HRP) mark is purchased from Beijing company of Zhong Shan Golden Bridge; O-Phenylene Diamine (OPD) is purchased from Harbin Bo Rui Bioisystech Co., Ltd; Dye in advance albumen Marker purchased from Fermentas company; SBA ClonotypingTM System/HRP Subclass of antibody identification kit is purchased from Southern Biotechnology company; Storage capacity is 2.7 * 10
9the Host Strains ER2738 of M13 phage random peptide library Ph.D.-12TM, tsiklomitsin (Tet) resistance all purchased from NEB company.
3. laboratory animal
6-8 BALB/c mouse in age in week is provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
The preparation of embodiment 1 monoclonal antibody
1. mouse immune
With BTV8(TCID
5010
8.25/ 0.1mL) 5 of totivirus immunity female BALB/c mouse in 6 weeks age, immunity is 3 times altogether, two weeks, each immune interval, immunizing dose is 0.5mL/, immunization route is peritoneal immunity.
Respectively two exempt to exempt from three after one week to the mouse blood sampling of docking, separation of serum (4 ℃, 10000rpm, 20min), detects antibody horizontal with indirect ELISA.In cytogamy first 3 days, the BALB/c mouse of good immune effect is carried out to booster immunization again, every mouse peritoneal injection 0.5mL immunizing antigen.
2. cytogamy
Merge and prepare feeder cell in first 1 day, according to ordinary method, get BALB/c mouse peritoneal macrophage and be laid in 96 porocyte culture plates stand-by.Disconnected neck is put to death the mouse of spleen to be got, and aseptic spleen the separating Morr. cell got carries out cytogamy in splenocyte and SP2/0 myeloma cell's ratio of 4: 1 with PEG, and the cell after fusion is laid on ready feeder cell.
3. the screening of positive hybridoma cell strain and clone
Utilize BTV8 to set up the strain of indirect ELISA detection method screening positive hybridoma cell, hybridoma enlarged culturing to reacting positive, with limiting dilution assay, carry out the subclone of positive hybridoma cell, at least subclone is 3 times simultaneously, and the positive hybridoma cell that subclone is good is frozen in time.The final hybridoma cell strain that obtains a strain can the anti-BTV-NS2 protein monoclonal antibody of stably excreting, its culture presevation number is: CGMCC No.7001; And by the monoclonal antibody called after BTV-4D4 of its secretion.
4. a large amount of preparations of monoclonal antibody
Give the healthy BALB/c mouse abdominal injection Freund's incomplete adjuvant about 10 week age, only, within 1 week, pneumoretroperitoneum injects 1 * 10 to 0.5mL/
6individual hybridoma extracts ascites after 7～10d when mouse web portion extreme expansion, every 2d, takes out once, by the centrifugal 10min of ascites 10000r/min extracting, removes upper strata grease and precipitation, and supernatant packing is stored in-20 ℃.
The evaluation of embodiment 2 monoclonal antibodies
1. the subgroup identification of monoclonal antibody
According to SBA ClonotypingTM System/HRP Subclass of antibody identification kit process specifications, the resulting monoclonal antibody of embodiment 1 is carried out to subgroup identification.
Result shows that the heavy chain of monoclonal antibody BTV-4D4 of the present invention is IgG1, and light chain is κ chain.IFA test-results shows that monoclonal antibody BTV-4D4 can react with 24 serotypes B TV.
2.Western blot test
Will be centrifugal cell precipitation after results BTV8 virus supernatant and BHK-21 cell precipitation carry out SDS-PAGE electrophoresis after processing, then through electric transfer printing by protein delivery to nitrocellulose filter, it is 18V voltage 30min that electricity turns condition; With 5% skim-milk confining liquid, 4 ℃ of sealings of the nitrocellulose filter after transfer printing are spent the night; Add monoclonal antibody supernatant incubated at room 1h, PBS damping fluid with PBST(containing the pH7.4 of 0.5mL/L tween 20) washing is three times, again with the goat dynamics incubated at room 1h of horseradish peroxidase (HRP) mark of 4000 times of dilutions, after PBST washing 3 times, with the colour developing of DAB colouring reagents box, sweep record.
Test-results confirms, the prepared monoclonal antibody BTV-4D4 of the present invention can with BTV-NS2 albumen generation specific reaction, and do not react (Fig. 1) with contrasting BHK-21 cell, according to this experimental result, infer that the BTV-NS2 protein B cell epitope that BTV-4D4 identifies should be a linear epitope simultaneously.
The evaluation of embodiment 3B cell epitope
1. the biopanning of Phage display random peptide library and phage genome sequencing
With reference to the random 12 peptide storehouse operational manuals of NEB company phage display, with the prepared monoclonal antibody BTV-4D4 being purified into, random peptide library is carried out to 3 and take turns elutriation, thereby elutriation goes out the phage of the displayed polypeptide Duan Keyu of institute monoclonal antibody specificity combination.3 take turns elutriation monoclonal antibody package amount is 100 μ g/mL, 150 μ L/ holes, and 3 take turns tween 20 concentration in elutriation PBST damping fluid used and be respectively 0.1%, 0.3% and 0.5%.
From the 3rd, take turns and the phage eluting, get 2 μ L and do 10 times of serial dilutions, each extent of dilution is got 10 μ L and is inoculated 200 μ L logarithmic phase ER2738 Host Strains, after effect 5min, whole bacterium liquid is mixed and is poured on the solid medium that contains IPTG and X-gal with 3mL top-agar substratum, 37 ℃ of overnight incubation.
Flat board from total amount less than 100 plaques, random 10 plaques of picking, inoculate respectively 8 pipe 1mL logarithmic phase ER2738 Host Strains, and 37 ℃ of shaking tables are cultivated 4.5h.Afterwards by culture 10000rpm, centrifugal 30s, supernatant moves in new pipe 10000rpm again, and centrifugal 30s, gets 600 μ L supernatants and is amplification phage storage liquid.According to the method for introducing in operational manual, can from above-mentioned phage storage liquid, extract phage genome DNA afterwards, and transfer to Shanghai Ying Jun Bioisystech Co., Ltd to carry out sequencing.
In 10 phage clones that the elutriation of phage sequencing result demonstration BTV-4D4 institute goes out, there are 4 to have some common sequences (Fig. 2), by relatively integrating, finally determine a concensus sequence SNYD, and on BTV-NS2 albumen, have this sequence, therefore think that SNYD should be the core sequence of the linear epitope identified of BTV-4D4.
The preliminary evaluation of 2.B cell epitope
The similar section of take on above-mentioned NS2 albumen is core, to albumen n end and C end, respectively extend 4～5 amino acid respectively, finally choosing length is that 13 amino acid whose small peptides (GVMRSNYDVRELR) are research object, carries out the preliminary evaluation of BTV-NS2 protein B cell epitope.Concrete grammar is as follows:
By Harbin plug, open up the synthetic above-mentioned small peptide of the biological agency of company limited, and the coated ELISA Sptting plate in small peptide 100ng/ hole is carried out to indirect ELISA detection to BTV-4D4.
Result shows that 13 amino acid short peptides of synthesized can specific reaction (Fig. 3) occur with BTV-4D4.And then the BTV-NS2 protein B cell epitope Primary Location that BTV-4D4 is identified in
146gVMRSNYDVRELR
158.
The accurate location of 3.B cell epitope
Utilize pepscan to synthesize following small peptide (table 1), and it is carried out to indirect ELISA detection, package amount is 100ng/ hole, and then accurately locates B cell epitope.
Table 1 is for the small peptide of BTV-4D4 synthesized
Result shows that P2, P3, P4, P6, P7, P8 and P9 all can be positive with BTV-4D4, and the OD492 value that P5 reacts with BTV-4D4 with P10 is lower, is about 0.2, illustrates that both joint efficiencies are lower (Fig. 3).The epi-position that the R that the above results explanation is 149 and the V of 154 identify BTV-4D4 is two key amino acids, their disappearance can significantly reduce the joint efficiency of BTV-4D4 and corresponding epi-position, the epi-position that other amino acid of disappearance are identified BTV-4D4, be some nonessential amino acid, their disappearance can not affect the joint efficiency of BTV-4D4 and corresponding epi-position.
In sum, the BTV-NS2 protein B cell epitope that the present invention identifies BTV-4D4 is accurately orientated as
149rSNYDV
154(shown in SEQ ID NO.1).
The conservative property of 4.B cell epitope and virus-specific analysis
Utilize European Bioinformatics Institute(EBI) the aminoacid sequence Blast program that provides of database, identified BTV-NS2 protein B cell epitope is carried out to conservative Analysis, whether this epitope sequences and other Orbivirus virus N S2 albumen respective section are compared, analyzing this epi-position is BTV specificity epitope simultaneously.
Blast result shows that the BTV-NS2 protein B cell epitope identifying is (Fig. 4) relatively guarding in each serotype strain of BTV, and higher with the NS2 albumen respective section homology of the epizootic hemorrhagic disease virus of deer (EHDV) of Orbivirus, and and other Orbivirus virus if AHSV, Chuzan virus NS2 albumen respective section are almost without homology (Fig. 4).
Claims (6)
1. the hybridoma cell strain of anti-blue tongue virus NS2 protein monoclonal antibody is secreted in a strain, and called after BTV-4D4 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation number is: CGMCC No.7001.
2. the monoclonal antibody that described in claim 1, hybridoma cell strain is secreted.
3. the linear B cell epitope polypeptide of blue tongue virus NS2 albumen, is characterized in that: the aminoacid sequence of described epitope polypeptide is that shown in SEQ ID NO.1, described epitope polypeptide is identified by monoclonal antibody claimed in claim 2.
4. the application of hybridoma cell strain in preparation diagnosis or prevention blue tongue virus infection medicine described in claim 1.
5. the application of monoclonal antibody in preparation diagnosis or prevention blue tongue virus infection medicine described in claim 2.
6. described in claim 3, the linear B cell epitope polypeptide of blue tongue virus NS2 albumen infects the application in reagent at preparation diagnosis blue tongue virus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219733A (en) * | 2015-10-14 | 2016-01-06 | 中国农业科学院哈尔滨兽医研究所 | The anti-monoclonal antibody BTV12-NS1-1F8 of blue tongue virus 12 type NS1 albumen and the B cell epi-position of identification thereof and application |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
| CN110016466A (en) * | 2019-05-21 | 2019-07-16 | 云南省畜牧兽医科学院 | The monoclonal antibody and its hybridoma cell strain of specific detection blue tongue virus and application |
-
2013
- 2013-10-14 CN CN201310478429.7A patent/CN103642757A/en active Pending
Non-Patent Citations (2)
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| YONG-LI QIN ET AL: "Identification of a linear B-cell epitope within the Bluetongue virus serotype 8 NS2 protein using a phage-displayed random peptide library", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》 * |
| 秦永丽: "抗蓝舌病病毒VP7和NS2蛋白单克隆抗体的制备及鉴定", 《中国预防兽医学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219733A (en) * | 2015-10-14 | 2016-01-06 | 中国农业科学院哈尔滨兽医研究所 | The anti-monoclonal antibody BTV12-NS1-1F8 of blue tongue virus 12 type NS1 albumen and the B cell epi-position of identification thereof and application |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
| CN106190986B (en) * | 2016-07-07 | 2019-05-17 | 东北农业大学 | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody |
| CN110016466A (en) * | 2019-05-21 | 2019-07-16 | 云南省畜牧兽医科学院 | The monoclonal antibody and its hybridoma cell strain of specific detection blue tongue virus and application |
| CN110016466B (en) * | 2019-05-21 | 2022-09-13 | 云南省畜牧兽医科学院 | Monoclonal antibody for specifically detecting bluetongue virus, hybridoma cell strain and application thereof |
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Inventor after: Wu Donglai Inventor after: Xu Qingyuan Inventor after: Sun Encheng Inventor after: Liu Nihong Inventor after: Yang Tao Inventor before: Wu Donglai Inventor before: Xu Qingyuan Inventor before: Liu Nihong Inventor before: Yang Tao |
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Application publication date: 20140319 |